HCV infections are a prevalent and growing health problem. It is estimated that as many as 2% of the US population, and 2.5% of the population world-wide, are infected by HCV. The disease currently causes 15,000 deaths/year in US alone, a number that is predicted to increase three-fold by 2010. Treatment options for HCV infected patients are quite limited, and none of the currently available treatments has a better than 50% probability of eliminating the infection from the patient. Consequently, there is a significant, and immediate unmet medial need for new and better drugs for the treatment of HCV infections. The cyclic peptides constitute a class of compounds that have made crucial contributions human health. These compounds have a considerable presence in several therapeutic areas, ranging from infectious diseases, to cancer and even autoimmune disorders. The discovery of the immunomodulatory cyclic peptide Cyclosporin A (CsA) 50 years ago had a truly profound impact and literally ushered in the era of modern transplantation medicine. Although cyclic peptides often are very efficient drugs, they are also complex natural product molecules (isolated from bacteria and fungi) and as such, they are difficult and expensive to synthesize and/or modify with conventional, synthetic chemistry-based methodologies. Consequently, currently used cyclic peptide-based drugs are either native compounds or native compounds with minor modifications. The vast majority of these compounds have not been optimized for human use and, consequently, the full potential of cyclic peptides, as human therapeutics, has not been explored. The overall goal of the project outlined in this proposal is to use a novel genetic engineering approach that allows cost-effective generation and production of both modified and new cyclic peptides, to generate new and improved anti-HCV drug candidates. The established immunomodulatory drug CsA, a compound with a wide range of pharmacological activities that includes anti-HCV activity, will be used as engineering template. The project involves development of methodologies and a set of genetic tools that allows introduction of modifications to the structure of native CsA by engineering the non-ribosomal peptide synthetase (NRPS) complex responsible its synthesis, in the producer organism Tolypocladium inflatum. Successful implementation of the envisioned genetic engineering approach will not only allow preparation of the envisioned novel anti-HCV drug candidate(s), but also compounds for other therapeutic applications, such as antifungal and antiparasitical compounds and perhaps even derivatives that retain the excellent immunomodulatory properties of native CsA, but not the nephrotoxicity. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) constitutes a significant and rapidly growing health problem world-wide. HCV infections are associated with considerable morbitity and mortality and existing therapies allows no more than a 50% probability of eliminating the virus form an infected patient. The principal aim of the proposed project is to use a novel genetic engineering approach to develop new, efficacious and well-tolerated drugs for the treatment of HCV infections. [unreadable] [unreadable] [unreadable]